Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS), the cause of which remains as yet unknown.
xe2x80x9cMultiple sclerosis (MS) is the most common neurological disease of young adults with a prevalence in Europe and North America of between 20 and 200 per 100,000. It is characterized clinically by a relapsing/remitting or chronic progressive course, frequently leading to severe disability. Current knowledge suggests that MS is associated with autoimmunity, that genetic background has an important influence and that xe2x80x9cinfectiousxe2x80x9d agent(s) may be involved. Indeed, many viruses have been proposed as possible candidates but as yet, none of them has been shown to play an aetiological role.
Many studies have supported the hypothesis of a viral aetiology of the disease, but none of the known viruses tested has proved to be the causal agent sought: a review of the viruses sought for several years in MS has been compiled by E. Norrby (1) and R. T. Johnson (2).
The discovery of pathogenic retroviruses in man (HTLVs and HIVs) was followed by great interest in their ability to impair the immune system and to provoke central nervous system inflammation and/or degeneration. In the case of HTLV-1, its association with a chronic inflammatory demyelinating disease in man (48) led to extensive investigations to search for an HTLV1-like retrovirus in MS patients. However, despite initial claims, the presence of HTLV-1 or HTLV-like retroviruses was not confirmed.
Recently, a retrovirus different from the known human retroviruses has been isolated in patients suffering from MS (3, 4, and 5).
In 1989, the authors described the production of extracellular virions, associated with reverse transcriptase (RT) activity, by a culture of leptomeningeal cells (LM7) obtained from the cerebrospinal fluid of a patient with MS (3). This was followed by similar findings in monocyte cultures from a series of MS patients (5). Neither viral particles nor viral RT-activity were found in control individuals. Furthermore, the authors were able to transfer the LM7 virus to non-infected leptomeningeal cells in vitro (26). The molecular characterization of the xe2x80x9cLM7xe2x80x9d retrovirus was a prerequisite for further evaluation of its possible role in MS. Considerable difficulties arose from the absence of continuously productive retroviral cultures and from the low levels of expression in the few transient cultures. The strategy described here focused on RNA from extracellular virions, in order to avoid non-specific detection of cellular RNA and of endogenous elements from contaminating human DNA. A specific retroviral sequence associated with virions produced by cell cultures from several MS patients has been identified. The entire sequence of this novel retroviral genome is currently being obtained using RT-PCR on RNA from extracellular virions. The retrovirus previously called xe2x80x9cLM7 virusxe2x80x9d corresponds to an oncovirus and is now designated MSRV (Multiple Sclerosis-associated RetroVirus).
The authors were also able to show that this retrovirus could be transmitted in vitro, that patients suffering from MS produced antibodies capable of recognizing proteins associated with the infection of leptomeningeal cells by this retrovirus, and that the expression of the latter could be strongly stimulated by the immediate-early genes of some herpesviruses (6).
All these results point to the role in MS of at least one unknown retrovirus or of a virus having reverse transcriptase activity which is detectable according to the method published by H. Perron (3) and qualified as xe2x80x9cLM7-like RTxe2x80x9d activity. The content of the publication identified by (3) is incorporated in the present description by reference.
Recently, the Applicant""s studies have enabled two continuous cell lines infected with natural isolates originating from two different patients suffering from MS to be obtained by a culture method as described in the document WO-A-93/20188, the content of which is incorporated in the present description by reference. These two lines, derived from human choroid plexus cells, designated LM7PC and PLI-2, were deposited with the ECACC on Jul. 22, 1992 and Jan. 8, 1993, respectively, under numbers 92072201 and 93010817, in accordance with the provisions of the Budapest Treaty. Moreover, the viral isolates possessing LM7-like RT activity were also deposited with the ECACC under the overall designation of xe2x80x9cstrainsxe2x80x9d. The xe2x80x9cstrainxe2x80x9d or isolate harboured by the PLI-2 line, designated POL-2, was deposited with the ECACC on Jul. 22, 1992 under No. V92072202. The xe2x80x9cstrainxe2x80x9d or isolate harboured by the LM7PC line, designated MS7PG, was deposited with the ECACC on Jan. 8, 1993 under No. V93010816.
Starting from the cultures and isolates mentioned above, characterized by biological and morphological criteria, the next step was to endeavour to characterize the nucleic acid material associated with the viral particles produced in these cultures.
The portions of the genome which have already been characterized have been used to develop tests for molecular detection of the viral genome and iunmunoserological tests, using the amino acid sequences encoded by the nucleotide sequences of the viral genome, in order to detect the immune response directed against epitopes associated with the infection and/or viral expression.
These tools have already enabled an association to be confirmed between MS and the expression of the sequences identified in the patents cited later. However, the viral system discovered by the Applicant is related to a complex retroviral system. In effect, the sequences to be found encapsidated in the extracellular viral particles produced by the different cultures of cells of patients suffering from MS show clearly that there is coencapsidation of retroviral genomes which are related but different from the xe2x80x9cwild-typexe2x80x9d retroviral genome which produces the infective viral particles. This phenomenon has been observed between replicative retroviruses and endogenous retroviruses belonging to the same family, or even heterologous retroviruses. The notion of endogenous retroviruses is very important in the context of our discovery since, in the case of MSRV-1, it has been observed that endogenous retroviral sequences comprising sequences homologous to the MSRV-1 genome exist in normal human DNA. The existence of endogenous retroviral elements (ERV) related to MSRV-1 by all or part of their genome explains the fact that the expression of the MSRV-1 retrovirus in human cells is able to interact with closely related endogenous sequences. These interactions are to be found in the case of pathogenic and/or infectious endogenous retroviruses (for example some ecotropic strains of the murine leukaemia virus), and in the case of exogenous retroviruses whose nucleotide sequence may be found partially or wholly, in the form of ERVs, in the host animal""s genome (e.g. mouse exogenous mammary tumor virus transmitted via the milk). These interactions consist mainly of (i) a trans-activation or coactivation of ERVs by the replicative retrovirus (ii) and xe2x80x9cillegitimatexe2x80x9d encapsidation of RNAs related to ERVS, or of ERVsxe2x80x94or even of cellular RNAsxe2x80x94simply possessing compatible encapsidation sequences, in the retroviral particles produced by the expression of the replicative strain, which are sometimes transmissible and sometimes with a pathogenicity of their own, and (iii) more or less substantial recombinations between the coencapsidated genomes, in particular in the phases of reverse transcription, which lead to the formation of hybrid genomes, which are sometimes transmissible and sometimes with a pathogenicity of their own.
Thus, (i) different sequences related to MSRV-1 have been found in the purified viral particles; (ii) molecular analysis of the different regions of the MSRV-1 retroviral genome should be carried out by systematically analyzing the coencapsidated, interfering and/or recombined sequences which are generated by the infection and/or expression of MSRV-1; furthermore, some clones may have defective sequence portions produced by the retroviral replication and template errors and/or errors of transcription of the reverse transcriptase; (iii) the families of sequences related to the same retroviral genomic region provide the means for an overall diagnostic detection which may be optimized by the identification of invariable regions among the clones expressed, and by the identification of reading frames responsible for the production of antigenic and/or pathogenic polypeptides which may be produced only by a portion, or even by just one, of the clones expressed, and, under these conditions, the systematic analysis of the clones expressed in the region of a given gene enables the frequency of variation and/or of recombination of the MSRV-1 genome in this region to be evaluated and the optimal sequences for the applications, in particular diagnostic applications, to be defined; (iv) the pathology caused by a retrovirus such as MSRV-1 may be a direct effect of its expression and of the proteins or peptides produced as a result thereof, but also an effect of the activation, the encapsidation or the recombination of related or heterologous genomes and of the proteins or peptides produced as a result thereof; thus, these genomes associated with the expression of and/or infection by MSRV-1 are an integral part of the potential pathogenicity of this virus, and hence constitute means of diagnostic detection and special therapeutic targets. Similarly, any agent associated with or cofactor of these interactions responsible for the pathogenesis in question, such as MSRV-2 or the gliotoxic factor which are described in the patent application published under No. FR-2,716,198, may participate in the development of an overall and very effective strategy for the diagnosis, prognosis, therapeutic monitoring and/or integrated therapy of MS in particular, but also of any other disease associated with the same agents.
In this context, a parallel discovery has been made in another autoimmune disease, rheumatoid arthritis (RA), which has been described in the French Patent Application filed under No. 95/02960. This discovery shows that, by applying methodological approaches similar to the ones which were used in the Applicant""s work on MS, it was possible to identify a retrovirus expressed in RA which shares the sequences described for MSRV-1 in MS, and also the coexistence of an associated MSRV-2 sequence also described in MS. As regards MSRV-1, the sequences detected in common in MS and RA relate to the pol and gag genes. In the current state of knowledge, it is possible to associate the gag and pol sequences described with the MSRV-1 strains expressed in these two diseases.
The present patent application relates to various results which are additional to those already protected by the following French Patent Applications:
No. 92/04322 of 03.04.1992, published under No. 2,689,519;
No. 92/13447 of 03.11.1992, published under No. 2,689,521;
No. 92/13443 of 03.11.1992, published under No. 2,689,520;
No. 94/01529 of 04.02.1994, published under No. 2,715,936;
No. 94/01531 of 04.02.1994, published under No. 2,715,939;
No. 94/01530 of 04.02.1994, published under No. 2,715,936;
No. 94/01532 of 04.02.1994, published under No. 2,715,937;
No. 94/14322 of 24.11.1994, published under No. 2,727,428;
and No. 94/15810 of 23.12.1994; published under No. 2,728,585.
The present invention relates, in the first place, to a viral material, in the isolated or purified state, which may be recognized or characterized in different ways:
its genome comprises a nucleotide sequence chosen from the group including the sequences SEQ ID NO:42, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:83, their complementary sequences and their equivalent sequences, in particular nucleotide sequences displaying, for any succession of 100 contiguous monomers, at least 50% and preferably at least 70% homology with the said sequences SEQ ID NO:42, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:83, respectively, and their complementary sequences;
the region of its genome comprising the env and pol genes and a portion of the gag gene, excluding the subregion having a sequence identical or equivalent to SEQ ID NO:1, codes for any polypeptide displaying, for any contiguous succession of at least 30 amino acids, at least 50% and preferably at least 70% homology with a peptide sequence encoded by any nucleotide sequence chosen from the group including SEQ ID NO:42, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:83 and their complementary sequences;
the pol gene comprises a nucleotide sequence partially or totally identical or equivalent to SEQ ID NO:53 or SEQ ID NO:87, excluding SEQ ID NO:1.
the gag gene comprises a nucleotide sequence partially or totally identical or equivalent to SEQ ID NO:82.
As indicated above, according to the present invention, the viral material as defined above is associated with MS. And as defined by reference to the pol or gag gene of MSRV-1, and more especially to the sequences SEQ ID NOS 47, 52, 53, 55, 56, 57, 82, 83, 87, 128, 129, 130, 131, 135, 136, 137 and 138, this viral material is associated with RA.
The present invention also relates to a nucleic material, in the isolated or purified state, having at least one of the following definitions:
a nucleic material comprising a nucleotide sequence selected from the group including sequences SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, their complementary sequences and their equivalent sequences, in particular nucleotide sequences displaying, for any succession of 100 contiguous monomers, at least 50% and preferably at least 60% homology with said sequences SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, and their complementary sequences, excluding HSERV-9 (or ERV-9); advantageously, the nucleotide sequence of said nucleic material is selected from the group including sequences SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, their complementary sequences and their equivalent sequences, in particular nucleotide sequences displaying, for any succession of 100 contiguous monomers, at least 70% and preferably at least 80% homology with said sequences SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, and their complementary sequences;
a nucleic material, in the isolated or purified state, coding for any polypeptide displaying, for any contiguous succession of at least 30 amino acids, at least 50%, preferably at least 60 %, and most preferably at least 70% homology with a peptide sequence encoded by any nucleotide sequence selected from the group including SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138 and their complementary sequences;
a nucleic material, in the isolated or purified state, of retroviral type, comprising a nucleotide sequence identical or similar to at least part of the pol gene of an isolated retrovirus associated with multiple sclerosis or rheumatoid arthritis; advantageously, said nucleotide sequence is 80% similar to said at least part of the gene pol;
a nucleic material comprising a nucleotide sequence identical or similar to at least part of the pol gen of an isolated virus encoding a reverse transcriptase having a enzymatic site comprised between the amino acid domains LPQG-YXDD, having a phylogenic distance with HSERV-9 of 0.063xc2x10.1, and preferably 0.063xc2x10.05; the phylogenic distances are calculated on the basis of a reference sequence according to UPGM tree option of the Geneworks(trademark) Software (INTELLIGENETICS); By enzymatic site, we understand the amino acids domain(s) conferring the specific activity of a given enzyme.
The present invention also relates to different nucleotide fragments each comprising a nucleotide sequence chosen from the group including:
(a) all the genomic sequences, partial and total, of the pol gene of the MSRV-1 virus, except for the total sequence of the nucleotide fragment defined by SEQ ID NO:1;
(b) all the genomic sequences, partial and total, of the env gene of MSRV-1;
(c) all the partial genomic sequences of the gag gene of MSRV-1;
(d) all the genomic sequences overlapping the pol gene and the env gene of the MSRV-1 virus, and overlapping the pol gene and the gag gene;
(e) all the sequences, partial and total, of a clone chosen from the group including the clones FBd3 (SEQ ID NO:42), t pol (SEQ ID NO:47), JLBc1 (SEQ ID NO:48), JLBc2 (SEQ ID NO:49) and GM3 (SEQ ID NO:52), FBd13 (SEQ ID NO:54), LB19 (SEQ ID NO:55), LTRGAG12 (SEQ ID NO:56), FP6 (SEQ ID NO:57), G+E+A (SEQ ID NO:83), excluding any nucleotide sequence identical to or lying within the sequence defined by SEQ ID NO:1;
(f) sequences complementary to the said genomic sequences;
(g) sequences equivalent to the said sequences (a) to (e), in particular nucleotide sequences displaying, for any succession of 100 contiguous monomers, at least 50% and preferably at least 70% homology with the said sequences (a) to (d), provided that this nucleotide fragment does not comprise or consist of the sequence ERV-9 as described in LA MANTIA et al. (18).
The term genomic sequences, partial or total, includes all sequences associated by coencapsidation or by coexpression, or recombined sequences.
Preferably, such a fragment comprises:
either a nucleotide sequence identical to a partial or total genomic sequence of the pol gene of the MSRV-1 virus, except for the total sequence of the nucleotide fragment defined by SEQ ID NO:1, or identical to any sequence equivalent to the said partial or total genomic sequence, in particular one which is homologous to the latter;
or a nucleotide sequence identical to a partial or total genomic sequence of the env gene of the MSRV-1 virus, or identical to any sequence complementary to the said nucleotide sequence, or identical to any sequence equivalent to the said nucleotide sequence, in particular one which is homologous to the latter.
In particular, the invention relates to a nucleotide fragment comprising a coding nucleotide sequence which is partially or totally identical to a nucleotide sequence chosen from the group including:
the nucleotide sequence defined by SEQ ID NO:36, SEQ ID NO:58 or SEQ ID NO:83;
sequences complementary to SEQ ID NO:36, SEQ ID NO:58 or SEQ ID NO:83;
sequences equivalent, and in particular homologous to SEQ ID NO:36, SEQ ID NO:58 or SEQ ID NO:83;
sequences coding for all or part of the peptide sequence defined by SEQ ID NO:35, SEQ ID NO:59 or SEQ ID NO:84;
sequences coding for all or part of a peptide sequence equivalent, in particular homologous to SEQ ID NO:35, SEQ ID NO:59 or SEQ ID NO:84, which is capable of being recognized by sera of patients infected with the MSRV-1 virus, or in whom the MSRV-1 virus has been reactivated.
The invention also relates to a nucleotide fragment (called fragment I) having at least one of the following definitions:
a nucleotide fragment comprising a nucleotide sequence selected from the group including SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, their complementary sequences, and their equivalent sequences, in particular nucleotide sequences displaying, for any succession of 100 contiguous monomers, at least 50% and preferably at least 60% homology with said sequences and their complementary sequences, said group excluding SEQ ID NO:1, said nucleotide fragment not comprising nor consisting of the sequence HSERV-9 (or ERV-9); preferably the nucleotide sequence of said fragment is selected from the group including SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, their complementary sequences, and their equivalent sequences, in particular nucleotide sequences displaying, for any succession of 100 contiguous monomers, at least 70% and preferably at least 80% homology with said sequences and their complementary sequences;
a nucleotide fragment comprising a coding nucleotide sequence which is partially or totally identical to a nucleotide sequence selected from the group including:
SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138; their complementary sequences; their equivalent sequences, in particular homologous to SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138;
sequences encoding all or parts of the peptide sequence defined by SEQ ID NO:89, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141;
sequences encoding all or parts of a peptide sequence equivalent, in particular homologous to SEQ ID NO:89, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, which is capable of being recognized by sera of patients infected with the MSRV-1 virus, or in whom the MSRV-1 virus has been reactivated.
The invention also relates to any nucleic acid probe for the detection of virus associated with MS and/or rheumatoid arthritis (RA), which is capable of hybridizing specifically with any fragment such as is defined above, belonging or lying within the genome of the said pathogenic agent. It relates, in addition, to any nucleic acid probe for detection of a pathogenic and/or infective agent associated with RA, which is capable of hybridizing specifically with any fragment as defined above by reference to the pol and gag genes, and especially with respect to the sequences SEQ ID NOS 36, 47, 52, 55, 56, 57, 58, 83 and SEQ ID NOS 35, 59 and 84.
The invention also relates to a primer for the amplification by polymerization of an RNA or a DNA of a viral material, associated with MS and/or RA, comprising a nucleotide sequence identical or equivalent to at least one portion of the nucleotide sequence of any fragment such as is defined above, in particular a nucleotide sequence displaying, for any succession of at least 10 contiguous monomers, preferably 15 contiguous monomers, more preferably 18 contiguous monomers and even most preferably 20 contiguous monomers, at least 70% homology with at least the said portion of the said fragment. Preferably, the nucleotide sequence of such a primer is identical to any one of the sequences selected from the group including SEQ ID NO:15 to SEQ ID NO:18, SEQ ID NO:43 to SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:93 to SEQ ID NO:99, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, and SEQ ID NO:145.
Generally speaking the invention also encompasses any RNA or DNA, and in particular replication vector, comprising a genomic fragment of the viral material such as is defined above, or a nucleotide fragment such as is defined above.
The invention also relates to the different peptides encoded by any open reading frame belonging to a nucleotide fragment such as is defined above, in particular any polypeptide, for example any oligopeptide forming or comprising an antigenic determinant recognized by sera of patients infected with the MSRV-1 virus and/or in whom the MSRV-1 virus has been reactivated. Preferably, this polypeptide is antigenic, and is encoded by the open reading frame beginning, in the 5xe2x80x2-3xe2x80x2 direction, at nucleotide 181 and ending at nucleotide 330 of SEQ ID NO:1.
The invention also encompasses the following polypeptides:
a)
a polypeptide encoded by any open reading frame belonging to a nucleotide fragment, fragment I, as defined above;
a polypeptide, characterized in that the open reading frame encoding it, is comprised, in the 5xe2x80x2-3xe2x80x2 direction, between nucleotide 18 and nucleotide 2304 of SEQ ID NO:87;
a polypeptide, having a peptide sequence comprising a sequence partially or totally identical to SEQ ID NO:89;
b)
a polypeptide, recombinant or synthetic, having a peptide sequence which comprises a sequence identical or equivalent to SEQ ID NO:90; in particular said polypeptide exhibits an enzymatic activity consisting of proteolytic activity;
a polypeptide, recombinant or synthetic, characterized in that the open reading frame encoding it begins, in the 5xe2x80x2-3xe2x80x2 direction, at nucleotide 18 and ends at nucleotide 340 of SEQ ID NO:87;
a polypeptide having an inhibitory activity on the proteolytic activity of a polypeptide as defined according to b);
c)
a polypeptide, recombinant or synthetic, having a peptide sequence which comprises a sequence identical or equivalent to SEQ ID NO:91; in particular said polypeptide exhibits a reverse transcriptase activity;
a polypeptide having a peptide sequence which comprises a sequence identical or equivalent to SEQ ID NO:92; in particular said polypeptide exhibits a ribonuclease activity;
a polypeptide, recombinant or synthetic, characterized in that the open reading frame encoding it begins, in the 5xe2x80x2-3xe2x80x2 direction, at nucleotide 341 and ends at nucleotide 2304 of SEQ ID NO:87;
a polypeptide, recombinant or synthetic, characterized in that the open reading frame encoding it begins, in the 5xe2x80x2-3xe2x80x2 direction, at nucleotide 1858 and ends at nucleotide 2304 of SEQ ID NO:87.
a polypeptide having an inhibitory activity on the reverse transcriptase activity of a polypeptide as defined according to c) or on the ribonuclease H activity of a polypeptide as defined according to c).
In particular, the invention relates to an antigenic polypeptide recognized by the sera of patients infected with the MSRV-1 virus, and/or in whom the MSRV-1 virus has been reactivated, whose peptide sequence is partially or totally identical or is equivalent to the sequence defined by SEQ ID NO:35, SEQ ID NO:59, SEQ ID NO:81, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:139, SEQ ID NO:140 and SEQ ID NO:141; such a sequence is identical, for example, to any sequence selected from the group including the sequences SEQ ID NO:37 to SEQ ID NO:40, SEQ ID NO:59 and SEQ ID NO:81.
The present invention also encompasses mono- or polyclonal antibodies directed against the MSRV-1 virus, which are obtained by the immunological reaction of a human or animal body or cells to an immunogenic agent consisting of an antigenic polypeptide such as is defined above.
The invention next relates to:
reagents for detection of the MSRV-virus, or of an exposure to the latter, comprising, at least one reactive substance selected from the group consisting of a probe of the present invention, a polypeptide, in particular an antigenic peptide, such as is defined above, or an anti-ligand, in particular an antibody to the said polypeptide;
all diagnostic, prophylactic or therapeutic compositions comprising one or more peptides, in particular antigenic peptides, such as are defined above, or one or more anti-ligands, in particular antibodies to the peptides, discussed above; such a composition is preferably, and by way of example, a vaccine composition.
The invention also relates to any diagnostic, prophylactic or therapeutic composition, in particular for inhibiting the expression of at least one virus associated with MS or RA, and/or the enzymatic activities of the proteins of said virus, comprising a nucleotide fragment such as is defined above or a polynucleotide, in particular oligonucleotide, whose sequence is partially identical to that of the said fragment, except for that of the fragment having the nucleotide sequence SEQ ID NO:1. Likewise, it relates to any diagnostic, prophylactic or therapeutic composition, in particular for inhibiting the expression of at least one pathogenic and/or infective agent associated with RA, comprising a nucleotide fragment such as is defined above by reference to the pol and gag genes, and especially with respect to the sequences SEQ ID NOS 36, 47, 52, 55, 56, 57, 58 and 83.
According to the invention, these same fragments or polynucleotides, in particular oligonucleotides, may participate in all suitable compositions for detecting, according to any suitable process or method, a pathological and/or infective agent associated with MS and with RA, respectively, in a biological sample. In such a process, an RNA and/or a DNA presumed to belong or originating from the said pathological and/or infective agent, and/or their complementary RNA and/or DNA, is/are brought into contact with such a composition.
The present invention also relates to any process for detecting the presence or exposure to such a pathological and/or infective agent, in a biological sample, by bringing this sample into contact with a peptide, in particular an antigenic peptide such as is defined above, or an anti-ligand, in particular an antibody to this peptide, such as is defined above.
In practice, and for example, a device for detection of the MSRV-1 virus comprises a reagent such as is defined above, supported by a solid support which is immunologically compatible with the reagent, and a means for bringing the biological sample, for example a sample of blood or of cerebrospinal fluid, likely to contain anti-MSRV-1 antibodies, into contact with this reagent under conditions permitting a possible immunological reaction, the foregoing items being accompanied by means for detecting the immune complex formed with this reagent.
Lastly, the invention also relates to the detection of anti-MSRV-1 antibodies in a biological sample, for example a sample of blood or of cerebrospinal fluid, according to which this sample is brought into contact with a reagent such as is defined above, consisting of an antibody, under conditions permitting their possible immunological reaction, and the presence of the immune complex thereby formed with the reagent is then detected.
Before describing the invention in detail, different terms used in the description and the claims are now defined:
strain or isolate is understood to mean any infective and/or pathogenic biological fraction containing, for example, viruses and/or bacteria and/or parasites, generating pathogenic and/or antigenic power, harbored by a culture or a living host; as an example, a viral strain according to the above definition can contain a coinfective agent, for example a pathogenic protist,
the term xe2x80x9cMSRVxe2x80x9d used in the present description denotes any pathogenic and/or infective agent associated with MS, in particular a viral species, the attenuated strains of the said viral species or the defective-interfering particles or particles containing coencapsidated genomes, or alternatively genomes recombined with a portion of the MSRV-1 genome, derived from this species. Viruses, and especially viruses containing RNA, are known to have a variability resulting, in particular, from relatively high rates of spontaneous mutation (7), which will be borne in mind below for defining the notion of equivalence,
human virus is understood to mean a virus capable of infecting, or of being harbored by human beings,
in view of all the natural or induced variations and/or recombination which may be encountered when implementing the present invention, the subjects of the latter, defined above and in the claims, have been expressed including the equivalents or derivatives of the different biological materials defined below, in particular of the homologous nucleotide or peptide sequences,
the variant of a virus or of a pathogenic and/or infective agent according to the invention comprises at least one antigen recognized by at least one antibody directed against at least one corresponding antigen of the said virus and/or said pathogenic and/or infective agent, and/or a genome any part of which is detected by at least one hybridization probe and/or at least one nucleotide amplification primer specific for the said virus and/or pathogenic and/or infective agent, such as, for example, for the MSRV-1 virus, the primers and probes having a nucleotide sequence chosen from SEQ ID NO:15 to SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:27 to SEQ ID NO:29, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, and their complementary sequences, under particular hybridization conditions well known to a person skilled in the art,
according to the invention, a nucleotide fragment or an oligonucleotide or polynucleotide is an arrangement of monomers, or a biopolymer, characterized by the informational sequence of the natural nucleic acids, which is capable of hybridizing with any other nucleotide fragment under predetermined conditions, it being possible for the arrangement to contain monomers of different chemical structures and to be obtained from a molecule of natural nucleic acid and/or by genetic recombination and/or by chemical synthesis; a nucleotide fragment may be identical to a genomic fragment of the MSRV-1 virus discussed in the present invention, in particular a gene of this virus, for example pol or env in the case of the said virus,
thus, a monomer can be a natural nucleotide of nucleic acid whose constituent elements are a sugar, a phosphate group and a nitrogenous base; in RNA the sugar is ribose, in DNA the sugar is 2-deoxyribose; depending on whether the nucleic acid is DNA or RNA, the nitrogenous base is chosen from adenine, guanine, uracil, cytosine and thymine; or the nucleotide can be modified in at least one of the three constituent elements; as an example, the modification can occur in the bases, generating modified bases such as inosine, 5-methyldeoxycytidine, deoxyuridine, 5-(dimethylamino)deoxyuridine, 2,6-diaminopurine, 5-bromodeoxyuridine and any other modified base promoting hybridization; in the sugar, the modification can consist of the replacement of at least one deoxyribose by a polyamide (8), and in the phosphate group, the modification can consist of its replacement by esters chosen, in particular, from diphosphate, alkyl- and arylphosphonate and phosphorothioate esters,
xe2x80x9cinformational sequencexe2x80x9d is understood to mean any ordered succession of monomers whose chemical nature and order in a reference direction constitute an item of functional information of the same quality as that of the natural nucleic acids,
hybridization is understood to mean the process during which, under suitable working conditions, two nucleotide fragments having sufficiently complementary sequences pair to form a complex structure, in particular double or triple, preferably in the form of a helix,
a probe comprises a nucleotide fragment synthesized chemically or obtained by digestion or enzymatic cleavage of a longer nucleotide fragment, comprising at least six monomers, advantageously from 10 to 1000 monomers, preferably 10 to 30 monomers and more preferably 18 to 30, and possessing a specificity of hybridization under particular conditions; preferably, a probe possessing fewer than 10 monomers, but preferably fewer than 15 monomers is not used alone, but is used in the presence of other probes of equally short size or otherwise; under certain special conditions, it may be useful to use probes of size greater than 100 monomers; a probe may be used, in particular, for diagnostic purposes, such molecules being, for example, capture and/or detection probes,
the capture probe may be immobilized on a solid support by any suitable means, that is to say directly or indirectly, for example by covalent bonding or passive adsorption,
the detection probe may be labelled by means of a label chosen, in particular, from radioactive isotopes, enzymes chosen, in particular, from peroxidase and alkaline phosphatase and those capable of hydrolyzing a chromogenic, fluorogenic or luminescent substrate, chromophoric chemical compounds, chromogenic, fluorogenic or luminescent compounds, nucleotide base analogues and biotin,
the probes used for diagnostic purposes of the invention may be employed in all known hybridization techniques, and in particular the techniques termed xe2x80x9cDOT-BLOTxe2x80x9d (9), xe2x80x9cSOUTHERN BLOTxe2x80x9d (10), xe2x80x9cNORTHERN BLOTxe2x80x9d, which is a technique identical to the xe2x80x9cSOUTHERN BLOTxe2x80x9d technique but which uses RNA as target, and the SANDWICH technique (11); advantageously, the SANDWICH technique is used in the present invention, comprising a specific capture probe and/or a specific detection probe, on the understanding that the capture probe and the detection probe must possess an at least partially different nucleotide sequence,
any probe according to the present invention can hybridize in vivo or in vitro with RNA and/or with DNA in order to block the phenomena of replication, in particular translation and/or transcription, and/or to degrade the said DNA and/or RNA,
a primer is a probe comprising at least six monomers, and advantageously from 10 to 30 monomers, and preferably from 18 to 25 monomers, possessing a specificity of hybridization under particular conditions for the initiation of an enzymatic polymeirzation, for example in an amplification technique such as PCR (polymerase chain reaction), in an elongation process such as sequencing, in a method of reverse transcription or the like,
two nucleotide or peptide sequences are termed equivalent or derived with respect to one another, or with respect to a reference sequence, if functionally the corresponding biopolymers can perform substantially the same role, without being identical, as regards the application or use in question, or in the technique in which they participate; two sequences are, in particular, equivalent if they are obtained as a result of natural variability, in particular spontaneous mutation of the species from which they have been identified, or induced variability, as are two homologous sequences, homology being defined below,
xe2x80x9cvariabilityxe2x80x9d is understood to mean any spontaneous or induced modification of a sequence, in particular by substitution and/or insertion and/or deletion of nucleotides and/or of nucleotide fragments, and/or extension and/or shortening of the sequence at one or both ends; an unnatural variability can result from the genetic engineering techniques used, for example the choice of synthesis primers, degenerate or otherwise, selected for amplifying a nucleic acid; this variability can manifest itself in modifications of any starting sequence, considered as reference, and capable of being expressed by a degree of homology relative to the said reference sequence,
homology characterizes the degree of identity of two nucleotide or peptide fragments compared; it is measured by the percentage identity which is determined, in particular, by direct comparison of nucleotide or peptide sequences, relative to reference nucleotide or peptide sequences,
this percentage identity has been specifically determined for the nucleotide fragments, clones in particular, dealt with in the present invention, which are homologous to the fragments identified, for the MSRV-1 virus, by SEQ ID NO:1 to NO:9, SEQ ID NO:42, SEQ ID NO:47 to SEQ ID NO:49, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:87, as well as for the probes and primers homologous to the probes and primers identified by SEQ ID NO:17 to SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:27 to SEQ ID NO:29, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:36, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:72, SEQ ID NO:76, and SEQ ID NO:93 to SEQ ID NO:99; as an example, the smallest percentage identity observed between the different general consensus sequences of nucleic acids obtained from fragments of MSRV-1 viral RNA, originating from the LM7PC and PLI-2 lines according to a protocol detailed later, is 67% in the region described in FIG. 1,
any nucleotide fragment is termed equivalent or derived from a reference fragment if it possesses a nucleotide sequence equivalent to the sequence of the reference fragment; according to the above definition, the following in particular are equivalent to a reference nucleotide fragment:
a) any fragment capable of hybridizing at least partially with the complement of the reference fragment,
b) any fragment whose alignment with the reference fragment results in the demonstration of a larger number of identical contiguous bases than with any other fragment originating from another taxonomic group,
c) any fragment resulting, or capable of resulting, from the natural variability of the species from which it is obtained,
d) any fragment capable of resulting from the genetic engineering techniques applied to the reference fragment,
e) any fragment containing at least eight contiguous nucleotides encoding a peptide which is homologous or identical to the peptide encoded by the reference fragment,
f) any fragment which is different from the reference fragment by insertion, deletion or substitution of at least one monomer, or extension or shortening at one or both of its ends; for example, any fragment corresponding to the reference fragment flanked at one or both of its ends by a nucleotide sequence not coding for a polypeptide,
polypeptide is understood to mean, in particular, any peptide of at least two amino acids, in particular an oligopeptide, or protein, and for example an enzyme, extracted, separated or substantially isolated or synthesized through human intervention, in particular those obtained by chemical synthesis or by expression in a recombinant organism,
polypeptide partially encoded by a nucleotide fragment is understood to mean a polypeptide possessing at least three amino acids encoded by at least nine contiguous monomers lying within the said nucleotide fragment,
an amino acid is termed analogous to another amino acid when their respective physicochemical properties, such as polarity, hydrophobicity and/or basicity and/or acidity and/or neutrality are substantially the same; thus, a leucine is analogous to an isoleucine.
any polypeptide is termed equivalent or derived from a reference polypeptide if the polypeptides compared have substantially the same properties, and in particular the same antigenic, immunological, enzymological and/or molecular recognition properties; the following in particular are equivalent to a reference polypeptide:
a) any polypeptide possessing a sequence in which at least one amino acid has been replaced by an analogous amino acid,
b) any polypeptide having an equivalent peptide sequence, obtained by natural or induced variation of the said reference polypeptide and/or of the nucleotide fragment coding for the said polypeptide,
c) a mimotope of the said reference polypeptide,
d) any polypeptide in whose sequence one or more amino acids of the L series are replaced by an amino acid of the D series, and vice versa,
e) any polypeptide into whose sequence a modification of the side chains of the amino acids has been introduced, such as, for example, an acetylation of the amine functions, a carboxylation of the thiol functions, an esterification of the carboxyl functions,
f) any polypeptide in whose sequence one or more peptide bonds have been modified, such as, for example, carba, retro, inverso, retro-inverso, reduced and methylenoxy bonds,
(g) any polypeptide at least one antigen of which is recognized by an antibody directed against a reference polypeptide,
the percentage identity characterizing the homology of two peptide fragments compared is, according to the present invention, at least 50% and preferably at least 70%.
In view of the fact that a virus possessing reverse transcriptase enzymatic activity may be genetically characterized equally well in RNA and in DNA form, both the viral DNA and RNA will be referred to for characterizing the sequences relating to a virus possessing such reverse transcriptase activity, termed MSRV-1 according to the present description.
The expressions of order used in the present description and the claims, such as xe2x80x9cfirst nucleotide sequencexe2x80x9d, are not adopted so as to express a particular order, but so as to define the invention more clearly.
Detection of a substance or agent is understood below to mean both an identification and a quantification, or a separation or isolation, of the said substance or said agent.